Ultrasound Modulates Calcium Activity in Cultured Neurons, Glial Cells, Endothelial Cells and Pericytes
Malachy Newman, Pratheepa Kumari Rasiah, Jiro Kusunose, Tonia S. Rex, Anita Mahadevan-Jansen, Jacob Hardenburger, E. Duco Jansen, Bryan Millis, Charles F. Caskey
Ultrasound in Medicine & Biology 2024, 50, 341-351 · 10.1016/j.ultrasmedbio.2023.11.004
Abstract
Objective Ultrasound is being researched as a method to modulate the brain. Studies of the interaction of sound with neurons support the hypothesis that mechanosensitive ion channels play an important role in ultrasound neuromodulation. The response of cells other than neurons (e.g., astrocytes, pericytes and endothelial cells) have not been fully characterized, despite playing an important role in brain function. Methods To address this gap in knowledge, we examined cultured murine primary cortical neurons, astrocytes, endothelial cells and pericytes in an in vitro widefield microscopy setup during application of a 500 ms burst of 250 kHz focused ultrasound over a pressure range known to elicit neuromodulation. We examined cell membrane health in response to a range of pulses and used optical calcium indicators in conjunction with pharmacological antagonists to selectively block different groups of thermo- and mechanosensitive ion channels known to be responsive to ultrasound. Results All cell types experienced an increase in calcium fluorescence in response to ultrasound. Gadolinium (Gad), 2-aminoethoxydiphenyl borate (2-APB) and ruthenium red (RR) reduced the percentage of responding neurons and magnitude of response. The percentage of astrocytes responding was significantly lowered only by Gad, whereas both 2-APB and Gad decreased the amplitude of the fluorescence response. 2-APB decreased the percentage of responding endothelial cells, whereas only Gad reduced the magnitude of responses. Pericytes exposed to RR or Gad were less likely to respond to stimulation. RR had no detectable effect on the magnitude of the pericyte responses while 2-APB and Gad significantly decreased the fluorescence intensity, despite not affecting the percentage responding. Conclusion Our study highlights the role of non-neuronal cells during FUS neuromodulation. All of the investigated cell types are sensitive to mechanical ultrasound stimulation and rely on mechanosensitive ion channels to undergo ultrasound neuromodulation.
Abstract via europepmc.
Exposures
Exposure 1: Primary rat cortical neurons, dosimetric pressure sweep
Target: cultured neurons — “Primary rat cortical neurons”
Device: Sonic Concepts · Sonic Concepts · H115 ✓
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 250 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | 100 | ✓✓✓ |
| Sonication duration (s) | 0.5 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable | |
| Pressure, domain unspecified (kPa) | 450, 900, 1,350swept | ✓✓✓ |
Cells were exposed to a single 500 ms burst with a 100% duty cycle. Exposures consisted of a single pulse with 125,000 cycles at 250 kHz.
Exposure 2: Primary rat cortical astrocytes, dosimetric pressure sweep
Target: cultured glia — “Primary rat cortical astrocytes”
Device: Sonic Concepts · Sonic Concepts · H115 ✓
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 250 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | 100 | ✓✓✓ |
| Sonication duration (s) | 0.5 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable | |
| Pressure, domain unspecified (kPa) | 450, 900, 1,350swept | ✓✓✓ |
Cells were exposed to a single 500 ms burst with a 100% duty cycle. Exposures consisted of a single pulse with 125,000 cycles at 250 kHz.
Exposure 3: Human brain cortex microvascular endothelial cells
Target: other — “Primary human brain cortex microvascular endothelial cells”
Device: Sonic Concepts · Sonic Concepts · H115 ✓
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 250 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | 100 | ✓✓✓ |
| Sonication duration (s) | 0.5 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable | |
| Pressure, domain unspecified (kPa) | 450 | ✓✓✓ |
Cells were exposed to a single 500 ms burst with a 100% duty cycle. Exposures consisted of a single pulse with 125,000 cycles at 250 kHz.
Exposure 4: Human pericytes
Target: other — “Primary human pericytes”
Device: Sonic Concepts · Sonic Concepts · H115 ✓
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 250 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | 100 | ✓✓✓ |
| Sonication duration (s) | 0.5 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable | |
| Pressure, domain unspecified (kPa) | 450 | ✓✓✓ |
Cells were exposed to a single 500 ms burst with a 100% duty cycle. Exposures consisted of a single pulse with 125,000 cycles at 250 kHz.
Flags from extraction
exposures[*].in_situ.pressure_kpa— Paper describes both free-field (open water) and in-situ (fiber-optic hydrophone in the dish, 1 mm above cells) pressure calibration, but does not explicitly state which calibration domain the reported 0.45/0.9/1.35 MPa exposure values correspond to; assigned to in_situ because the fiber-optic hydrophone was 'selected for in situ measurements' of pressure 'within the dish' where the cells sit.n_subjects— Paper reports cell counts per group (e.g., N > 200 to N > 5000 cells) but never the number of independent cultures/dishes/biological replicates used.exposures[2].target.terms— Endothelial cells have no matching target id in the controlled vocabulary; classified as 'other'.exposures[3].target.terms— Pericytes have no matching target id in the controlled vocabulary; classified as 'other'.sham_type— Paper refers to 'sham exposures' / 'sham-exposed' cells (e.g., for endothelial cells and pericytes) without describing the sham mechanism (e.g., transducer off vs blocked); classified as 'other'.